Abstract Staphylococcus epidermidis (SE) ranks as one of the most common causes of nosocomial infections in US hospitals. Intravascular devices and prostheses are the usual sites of infection followed by seeding of the bloodstream. Although more indolent than S. aureus, SE infections are difficult to treat due to its persistence as biofilm on abiotic surfaces as well as its penchant for antibiotic resistance. There are two major issues in advancing our understanding of SE pathogenesis: 1) prior work has focused on two laboratory isolates, SE 1457 and RP62A while our knowledge on clinical isolates are virtually non-existent; 2) SE clinical isolates are extremely difficult to transform due to its formidable restriction barrier. As a consequence, we lack the ability to manipulate clinical isolates genetically to dissect the relevant pathogenic steps leading to colonization, biofilm formation, persistence and dissemination. With the advance of genomics and SMRT sequencing, we are in a unique position to analyze the methylation pattern of the restriction modification (RM) system based on the specificity unit (HsdS) and methylase (HsdM). There are four RM systems in SE (types I-IV). Type 1 and type IV are the major restriction barriers that prevent uptake of foreign DNA due to restriction by HsdR. We will exploit the RM system by methylating adenine in plasmid DNA with type I cognate HsdMS complex(es) in E. coli DC10B which is defective in cytosine methylation and can bypass the type IV RM system. While type IV RM system is conserved among S. aureus and SE, the type I RM system is more variable and entails at least 7 distinct groups, each with its unique target recognition motif (TRM) mediated by HsdS. These discrepancies help explain the differences in restriction among SE isolates. We hypothesize that type 1 RM system in a particular SE isolate can be completely bypassed to yield efficient transformation by cloning shuttle plasmids in DC10B that express the cognate hsdMS genes. With this goal is mind, we propose the following two specific aims: I) determine the different groups in type I RM system by performing Pacbio single molecule real time sequencing (SMRT) to detect methylated-adenine residues and the target recognition motif of the HsdMS complex in the SE genomes; II) Construction of DC10B-derived E. coli strains to enable efficient transformation into diverse SE strains. As a utility from this system, we will construct two relevant mariner transposon libraries to be available to the research community.